Allostery and Cellular Distribution of the Nitric Oxide Recepto, Soluble...

摘要： 一氧化氮（NO）是一种小的活性分子，参与许多信号传导途径，包括 调节血管生成和转移。一氧化氮的主要细胞受体是可溶性鸟苷酸环化酶 (sGC)，一种150 kDa的异二聚体血红素蛋白，是治疗疾病的有吸引力的靶点， 包括癌症NO的结合刺激sGC活性，导致cGMP信号传导的建立。 级联。sGC受多种分子的变构调节，包括NO、ATP、YC-1（一种小的 核苷酸样药效团）和通过翻译后修饰。尽管进行了广泛的研究， 已知sGC的整体形状，变构调节发生的方式， 蛋白质中功能结构域的排列或蛋白质在细胞中的排列。我们打算 通过基于荧光的方法填补这一空白，使我们能够测量内部的结构变化， sGC，并且还监测细胞内的sGC定位。具体来说，我们打算将配对 荧光团的全长和截断形式的sGC，使得FRET测量将揭示 刺激和抑制条件下功能域之间的距离。我们已经开发出一种 涉及来自天蛾（Manducasexta）的sGC的稳健模型系统，用该系统开始这些研究， 但一旦建立荧光系统，也将包括人sGC。我们会调查sGC 构象状态不仅与孤立的材料，而且在活细胞。此外，我们将使用 免疫组织学和荧光显微镜的组合以监测活化下sGC的定位 和抑制条件。我们已经表明，sGC在细胞中显示点状排列，但 这种明显的模式的功能后果是未知的。总之，我们希望这些研究将 揭示sGC经历的结构转变，并为药物治疗的新策略提供框架 的发现这一试点项目产生的成果将为今后的合作供资奠定基础 和研究工作。

ABSTRACT: Nitric oxide (NO) is a small, reactive molecule involved in numerous signaling pathways, including those regulating angiogenesis and metastasis. The primary cellular receptor for NO is soluble Guanylyl Cyclase (sGC), a heterodimeric hemoprotein of 150 kDa and an attractive target for the treatment of disease, including cancer. Binding of NO stimulates sGC activity, leading to the establishment of a cGMP signaling cascade. sGC is allosterically regulated by a variety of molecules, including NO, ATP, YC-1 (a small nucleotide-like pharmacophore), and by posttranslational modifications. Despite extensive study, little is known about the overall shape of sGC, the means by which allosteric regulation takes place, the arrangement of functional domains in the protein or the arrangement of the protein in the cell. We intend to fill this gap through fluorescence-based approaches that will allow us to measure structural changes within sGC, and also to monitor sGC localization within the cell. Specifically, we intend to incorporate paired fluorophores to full-length and truncated forms of sGC such that FRET measurements will reveal the distances between functional domains under stimulating and inhibiting conditions. We have developed a robust model system involving sGC from the hawkmoth {Manduca sexta) with which to begin these studies, but will also include human sGC once the fluorescence system is established. We will investigate sGC conformational states not only with isolated material, but also in live cells. Additionally, we will use a combination of immunohistology and fluorescence microscopy to monitor localization of sGC under activating and inhibiting conditions. We have shown that sGC displays a punctate arrangement in the cell, but the functional consequences of this stark pattern are unknown. Together, we expect that these studies will uncover the structural transitions that sGC undergoes and provide the framework for novel strategies in drug discovery. The generated results from this pilot project will provide the basis for future collaborative funding and research efforts.